PROJECT SUMMARY Failure of new drugs at late stages of development is an extremely costly event, commonly associated with the detection of unexpected arrhythmogenic properties in novel drugs. Undetected arrhythmia-inducing effects are also a common reason for drug withdrawal from the market. As a result, the FDA now mandates that all new drugs be tested for potential arrhythmogenic properties, which has led to a growing market for accurate and cost effective preclinical screening tools. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer the means to generate superior in vitro cardiac tissues for such applications. However, an inability for hPSC-CMs to develop into adequate representations of adult myocardial tissue under standard culture conditions is a major impediment to the use of such cell-constructs in effective preclinical screening protocols. Generation of mature cardiac tissues that accurately recapitulate the form and function of the adult human heart is necessary to provide preclinical data capable of reliably predicting a compound?s efficacy and/or toxicity when transferred to a clinical setting. Our Phase 1 SBIR project demonstrated that nanopatterned microelectrode arrays (nanoMEAs) can be used to promote cardiomyocyte maturation to the point where more representative drug responses are achieved. Based on results achieved during Phase 1, NanoSurface Biomedical is applying for Phase 2 SBIR funding to further develop and optimize an integrated prototype nanoMEA system to enhance cardiac maturation and high throughput functional analysis for improved drug-induced cardiotoxicity screening. We hypothesize that the establishment of a 384-well nanoMEA plate will improve cardiac structural and functional development to enable the collection of high throughput compound toxicity data with greater predictive capacity. To test these hypotheses, this grant renewal will focus on the validation of our optimized high throughput nanoMEA plate design, including the establishment of custom-built hardware and software to facilitate rapid data analysis as well as development of key biological metrics for device validation (Aim 1). We will then use this platform to investigate the ability for hPSC-CMs to generate functional responses to known arrhythmogenic compounds that are representative of these drugs? activity in vivo (Aim 2). Cell line variability will also be investigated to understand how genotypic differences translate into functional variance in vitro. Lastly, the capacity for nanopatterned MEAs to promote the development of disease phenotypes in hPSC-CMs from structural cardiomyopathy patients will be investigated as a means to broaden the utility of our eventual product. The structural impact of nanopatterns on cardiomyocytes provides strong rationale for the ability for these topographic substrates to help stratify disease phenotypes from wild type controls, and represents a second substantial market for this technology. Successful validation of our high throughput nanopatterned MEA system will produce an innovative new product designed specifically to relieve critical deficiencies and reduce cost in the current preclinical drug development process.